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IMPACT OF ENTERIC INFECTIONS ON COGNITIVE DEVELOPMENT: FIELD AND ANIMAL STUDIES OF

PROTECTION BY ApoE4

RICHARD L. GUERRANT1, REBECCA J. SCHARF1, ORLEÁNCIO AZEVEDO2, LUTHER BARTELT1, ALDO A.M. LIMA2,

and REINALDO B. ORIÁ2

1Center for Global Health, Division of Infectious Diseases and International Health, Departments of Medicine and Pediatrics, University of Virginia,

Charlottesville, USA, and 2Center for Global Health, Department of Physiology and Pharmacology, INCT-Biomedicine, Faculty of Medicine,

Federal University of Ceará, Ceará, Brazil

SUMMARY

Although diarrhoeal diseases in early childhood remain important causes of mortality, the long-term impact of enteric infections, with or without overt diarrhoea, may impair child growth and development. Repeated dehydrating or malnourishing infections can compound other causes of mortality and may also have lifelong consequences for those who survive. This includes a cognitive decrement that may average up to 10 IQ points by 7-9 years of age that can be attributed to diarrhoeal illnesses in the first 2 years of life alone. The cognitive function most affected is semantic, rather than phonemic, fluency, a deficit also seen in early Alzheimer’s (vs. Parkinson’s) dementia. These effects may be lifelong and may have selected for surprising “sur-vival” or “thrift” genetic alleles like ApoE4 that we have shown to protect against the cognitive deficits seen in children with heavy diarrhoea burdens as well as providing protection against the growth and histopathologic im-pact of enteric infections in a murine model of malnutrition and infection.

In this overview, we review these long-term growth and cognitive effects of early childhood diarrhoea, the Alzheimer-like deficits seen and the surprising protection provided by the ApoE4 allele that increases Alzheimer risk in later life, a new example of “antagonistic pleotropy”. We also show this protection in our murine model of cryptosporidiosis using targeted transgenic mice with the human E4 allele and how the molecular mecha-nism of this benefit can lead to novel interventions to break the vicious cycle of diarrhoea and malnutrition and their devastating consequences for child development.

BETTER COGNITION AND IQ CORRELATE WITH FEWER INFECTIONS

Over 200 million of the world’s chil-dren less than 5 years of age fail to achieve their developmental potential, are stunted and live in poverty. These children do poorly in school and go on

to have low incomes, high fertility and provide poor care for their children, contributing to what Dr. Grantham-McGregor has called “intergenerational transmission of poverty” (Grantham-

Old Herborn University Seminar Monograph 26: The gut microbiome and the nervous system. Editors: Peter J. Heidt, John Bienenstock, and Volker Rusch. Old Herborn University Foundation, Herborn-Dill, Germany: 105-116 (2013).

McGregor et al., 2007). Eppig and col-leagues recently suggested that the “Flynn Effect” of improving IQ with development correlates with decreasing “infectious diseases burden”, even when controlling for GDP per capita, education, temperature and malnutri-tion; i.e. the recognized correlation of nutritional deficiencies with IQ is lost when the effects of infectious diseases are removed which they interpret as suggesting that the malnutrition-IQ link

appears to occur through infectious dis-eases (Eppig et al., 2010). Whether there is an effect of infections on cog-nitive development that is independent of their effects on malnutrition is less clear. Although this is a controversial area, our data on diarrhoeal illness cor-relations also appear to be independent of stunting (<1HAZ) (which is itself also correlated with diarrhoea in the first 2 years of life) (Pinkerton et al., 2012).

INFECTION OR STUNTING IMPAIRS COGNITION

Convincing support for the effects of infection on child development came from albendazole trials in Kenya and Jamaica, suggesting that intestinal hel-minths impair growth and cognitivedevelopment. These were the basis of our studies of heavy early childhood diarrhoea burdens in Fortaleza. Ste-phenson and colleagues reported that, among Kenyan schoolchildren, even a single dose of albendazole showed a benefit in fitness, appetite and weight and height gains within 2 to 4 months when compared with double blinded placebo-treated controls (Stephenson et al., 1989, 1990, 1993). Similarly, in studies of schoolchildren in Jamaica, Nokes et al. (1992) showed improved fluency (long term memory and re-trieval) and digit span back-wards/forwards (from WISC, involves attention and distractibility) 2 months after a 3-day course of albendazole (vs.placebo). Since HAZ-2 may be a surro-gate for early childhood diarrhoea bur-dens (Dillingham and Guerrant, 2004),it may also be relevant that Chang and co-workers described better arithmetic scores at 10 years old with higherHAZ-2 (height for age Z scores at 2 years old) (Chang et al., 2011). Alt-hough these brief anti-helminthic treat-ments did not necessarily eradicate in-testinal geohelminths (and certainly did

not prevent common re-infections), the2-3 log reduction in major geohel-minths provide impressive evidence for the importance of heavy intestinal nematode infections in the physical and cognitive development of schoolchil-dren. Our own data from Northeast Brazil also suggests that geohelminth infections in early childhood (i.e. from birth to 2 years old) have important, lasting effects on subsequent child growth and development (Moore et al., 2001). We and others are now explor-ing whether stunted children may also be at greater risk for later obesity, dia-betes and metabolic syndrome in what we have called a ‘collision of bad water with bad diets’ (DeBoer et al., 2012).

The importance of stunting on cog-nitive development is clear from sev-eral studies (Grantham-McGregor, 1995). In studies of nonverbal intelli-gence at 8-11 years old in the Philip-pines, Mendez and Adair’s group has shown that moderate to severe stunting (i.e. HAZ-2 <-2) at 2 years is associ-ated with a 0.25 to 0.61 standard devia-tion lower mean cognitive test Z score by age 8 (p<0.001) (Mendez and Adair, 1999). They also noted that stunting (i.e. progressing from -1.5 to -3.3 HAZ) at 2 years correlated with pro-gressive delays in age at starting school from 5 to 8+ years, and that, even

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Figure 1: Age specific diarrhoea attack rates in rural and urban communities (Pacatuba andGoncalves Dias) in Northeast Brazil (Guerrant et al., 1983; Lima et al., 2000).

though schooling (achieving 2-6 years rement in the stunted children at 11 of education) improves mean cognitive years; i.e. stunting limits what educa-test scores, there remains a 30% dec- tion can accomplish!

THE CRITICAL 4-24 MONTH AGE “WINDOW” FOR CHILD DEVELOPMENT

Leonardo Mata, in studying The Chil-dren of Santa Maria Cauque (Mata, 1978) described how even impover-ished children often start off on their growth curves reasonably well, only to fall progressively off with the onset of diarrhoeal and other common earlychildhood infections over the critical first 2 years of life. Precisely the same pattern of fall-off in linear growth (i.e. HAZ) occurs worldwide, with remarka-bly similar findings in Asia, Africa and Latin America described by Victora and Shrimpton (Victora et al., 2010) in 2010, much as they had shown nearly a decade earlier in 2001 (Shrimpton et al., 2001).

Feeding studies also confirm the importance of early childhood (3 years) in cognitive development. For example, in the two Guatemalan villages given an “atole” vegetable-protein supple-ment (163kcal with 11.5g protein) vs. “fresco” sugar sweetened beverage(59kcal and no protein) in two control villages in INCAP studies from 1969-1977. A follow-up 25-35 years later showed that those in the supplemented villages had a 10% better IQ, even af-ter, adjusting for schooling (Stein et al., 2008), and that the 25-42 year old males had 46% higher wages, with the females having 8-20% higher reading, Raven scores and schooling if they had

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been in the supplemented villages com-pared with the non-supplemented vil-lages some 40 years before. However these benefits were seen only in those who had been in the supplement pro-gram in their first 2-3 years of life, showing the crucial timing of early life in cognitive development (Hoddinott et al., 2008).

We also have found increasingly impressive correlations in our Fortaleza studies of early childhood diarrhoea (ECD) that heavy diarrhoea burdens in the first two years of life are associated with impairments in cognitive function (TONI, WISC-III coding and digit span and WRAML mazes) several yearslater (Guerrant et al., 1999; Niehaus et al., 2002). Furthermore, these heavy diarrhoea burdens in the first 2 years of life also correlate with impairedschooling (both age at starting school and age-four-grade) several years later (Lorntz et al. 2006). Early heavy diar-rhoea burdens are also associated with stunting at 2 years of age in our as well as in other multi-country studies (Moore et al. 2001; Checkley et al., 2008) and, as noted above, stunting at the second birthday (HAZ-2) is clearly associated with impaired cognition as assessed later in life. Although some suggest that the effects of diarrhoea on cognition may be only through its ef-fects on stunting, as noted above, like Eppig, we find that the association of diarrhoea with cognitive impairment remains even when controlling for an-thropometry. Thus heavy early child-hood diarrhoea burdens clearly associ-ate with lasting effects on stunted growth, and, in turn on impaired cogni-tion. Whether the cognitive impact of early childhood diarrhoea is independ-ent of (i.e. even greater than) the also significant effects of diarrhoea on stunting remains controversial. Never-theless, the huge effects of early child-hood diarrhoea on child growth and

development are critical to recognize and ameliorate.

The reasons for the first 2 years of life being so critical are likely at least two-fold: first is the obvious vulnera-bility to the heaviest burdens of diar-rhoea and enteric infection (with rates clearly highest in that age range)(Guerrant et al., 1983; Lima et al., 2000; Fischer Walker et al., 2012)(Figure 1) Second is the rapid develop-ment of brain growth and synaptogene-sis and myelination in humans in this window from birth to 2 years. Unlike some other species in which brain de-velopment occurs predominantly in utero, human infants are born with small brains and sparse synapses rela-tive to their near adult levels of brain weight-for-height and even synaptic density by 2 years of life (Dobbing and Sands, 1973; Corel, 1975; Rice and Barone, 2000; Thompson and Nelson, 2001) (Figure 2). Not only does the human brain double in size in the first year of life, by 3 years of age it reaches 80% of its adult volume (The Urban Child Institute: Baby's brain begins now, Conception to age 3. URL: www.theurbanchildinstitute.org/why-0-3/baby-and-brain, 2012; Nowakowski, 2006). Furthermore, synapse formation occurs predominantly between birth and 2-3 years of age (by which time the human brain has nearly 200% of its adult number of synapses); after which “blooming and pruning” graduallyeliminate nearly half of these synapses throughout childhood and adolescence (Corel, 1975; Huttenlocher, 2002).These anatomic observations are even more apparent when one considers the impressive incapacity of the human new-born infant compared with the remarkable appearance of motor and cognitive skills including walking, running, talking and personality which develop by the child’s second birthday.

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Figure 2: Age of brain development in humans and other animals, showing that most human brain development (by A: weight or by B: synapse formation) occur in the first 2 years of life. Adaptedrespectively from Dobbing and Sands (1973) and from Corel (1975).

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Figure 3: Brain regional development and function.

EARLY CHILDHOOD DIARRHOEA AND GIARDIASIS IMPAIR SEMANTIC FLUENCY (LIKE ALZHEIMER’S DISEASE)

AND ApoE4 PROTECTS

When we examined the specific areas Because of the semantic fluency most affected by early childhood diar- predominance of the deficits, we exam-rhoea, it was striking that these were ined the ApoE4 allele frequencies and predominantly functions similar to found, to our initial surprise, that those lost in early Alzheimer’s disease. ApoE4 was protective of the cognitive These include higher executive func- function and against diarrhoea, the tion and semantic (vs. phonetic) flu- former only in the children with heavy ency (Patrick et al., 2005; Oria et al. diarrhoea burdens (Oria et al., 2005, 2009), with brain regions that likely 2007, 2010). Some early investigations differ from the predominant ones in- into this link are beginning to support a volved in phonemic fluency or in Par- host benefit of ApoE4 against enteric kinson’s dementia (Figure 3). protozoa. The positive correlation

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between Giardia lamblia and diarrhoea (p<0.01) in ApoE4 negative children in our study was lost in the presence of an ApoE4 allele (p=0.53) (Oria et al., 2005). Moreover, the ApoE4 allele appears to be protective againstcognitive impairments due to earlyGiardia infections in Egypt (Yahya et al., 2009a, 2009b), although these findings should be further confirmed with larger numbers and better control for confounding factors. The association of potential protectionagainst symptoms with this parasite by the host ApoE4 allele may be related to the Giardia’s requirement to obtain host cholesterol for its own growth(since Giardia is unable to synthesize cholesterol) thus needing to divert host cholesterol to the parasite from the in-testinal milieu, which may deleteri-ously affect the developing brain (Oria et al., 2007). These effects may require an evolutionarily conserved LDL-re-ceptor pathway, which is found to be down-regulated in ApoE4 carriers (Mahley and Rall, 2000). Recently, a putative Giardia lamblia low-density lipoprotein receptor-related protein(GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and an FXNPXY-type endocytic motif with human LRPs, was identified (Mahley and Rall, 2000; Rivero et al., 2011).

In addition, Colton and colleagues (Colton et al., 2001; Czapiga and Colton, 2003) have shown that ApoE4 increases NO production in microglial macrophages by stimulating arginine uptake via an arginine-selective cati-onic amino acid transporter (CAT1). Hence we examined the ability of argi-nine to enhance parasite killing with Cryptosporidium infections in our mu-rine model (Castro et al., 2012) and also the ability of targeted transgenic C57Bl6 mice with the human ApoE4 allele to resist cryptosporidial infec-

tions or their growth impairment in our murine model. We found that arginine is the most effective “anticryptosporid-ial drug” we have encountered in our murine model, with a 10-fold reduction in the number of parasites per milli-gram of intestinal tissue. These effects were only partially blocked by L-NAME (NG-nitro-arginine methyl es-ter) (Castro et al. 2012) or by BEC (S-(2-boronoethyl)-L-cysteine) (Castro et al., unpublished data), suggesting that the protection from arginine was through both the iNOS and the arginase pathways to kill the parasite and repair epithelial cell injury respectively. Fur-thermore, in studies that are beingsubmitted for publication elsewhere, C57BK6J ApoEko mice expressing the human ApoE4/4 gene under murine ApoE promoter lost less weight, had better villus-to-crypt ratios and shed fewer parasites than ApoE 3/3 target replacement and wild-type mice, suggesting that understanding how ApoE4 may be protective can opennovel approaches to better controlling protozoal infections. Interestingly, sup-porting the concept that ApoE4 may improve innate immunity against en-teric pathogens, data from the Tsimane population in lowland Bolivia, (an in-digenous forager-farmer populationliving under conditions resembling pre-industrial European populations with high infectious morbidity, high infec-tion and inflammation, and shortened life expectancy) when bearing ApoE4 show lower serum C-reactive protein levels (Vasunilashorn et al., 2011), suggesting that ApoE4 bearers had lower rates of environmental-related infections.

Our data support that ApoE4 be-haves as antagonistic pleiotropy, where the presence of ApoE4 in our gene pool was associated with increased needs for fat energy storage and improved innate immunity adaptations against enteric

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infections in times where those were critical for human survival in the pre-industrialized era. However this once beneficial gene becomes potentiallyquite detrimental in the presence of longer life expectancy, reduced physi-cal exertion and westernized diets.

There are other postulates and literature evidence for an antagonistic pleiotropy described for ApoE4 during human early development and in adulthood (Prentice et al., 2005; Alexander et al., 2007; Finch and Morgan, 2007; Beeri et al., 2009; Chang et al., 2011).

EFFECTS OF MALNUTRITION AND OF MICRONUTRIENTS ON BRAIN ANATOMY AND FUNCTION

Especially vulnerable in the first two years of life in humans (analogous to the first few weeks of life in rodent models) is the postnatal brain plasticity, particularly in dynamic hippocampal, neocortical and cerebellar regions(Frankova and Barnes, 1968; Rice and Barone, 2000). It is these areas that are potentially altered by nutrients or micronutrients in critical developmen-tal windows (Georgieff, 2007; Pinero et al., 2001; de Souza et al., 2011).Hence we examined the effects of mal-nutrition and of zinc and glutamine therapy in our murine model of litter clustering induced malnutrition. There we found that malnourished mice (with breast-milk restriction by increased lit-ter size) had growth impairment and deficits in early post-natal behaviour ontogeny, associated with reduced se-rum and brain zinc levels. In addition, we found reduced hipocampal GABA levels and reductions in hippocampal synaptophysin (as a marker for synap-tic density) expression, associated with malnutrition. Furthermore, malnutri-tion-induced CA-1 neuronal hypertro-phy was found with litter size cluster-ing, likely related to CA-1 cell death changes. All effects improved with glu-

tamine and/or zinc treatment (Ladd et al., 2010).

We next examined the effects of zinc, vitamin A and glutamine supple-mentation on the growth and cognitive responses in 213 undernourished chil-dren from the favela in Fortaleza and found that lower vitamin A and gluta-mine levels were associated with dis-rupted intestinal barrier function (by lactulose/mannitol absorption ratios). There was also a significant correlation between vitamin A supplementation of apolipoprotein E4(+) children and im-proved lactulose/mannitol absorptionratios. In addition, only the ApoE4positive children (37/213, 13.9%) who received glutamine supplementation(10 day glutamine treatment either with or without zinc or vitamin A supple-mentation) showed significant positive Pearson correlations between the changes in height-for-age z-scores over four months with delayed verbal learn-ing scores, along with correlated changes over the same period in weight-for-age z-scores and weight-for-height z-scores that were associated with non-verbal intelligence quotients (Mitter et al., 2012).

CONCLUSIONS

In conclusion, we find profound and dren’s growth and cognitive develop-lasting effects of early childhood en- ment. While these “vicious cycles of teric infections and diarrhoea on chil- poverty” are only now being dissected

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at the levels of molecular causality, ge- ing these vicious cycles of diarrhoea, netic predispositions and potential stunting, cognitive impairment and novel interventions can already be de- poverty. signed that hold promise for interrupt-

ACKNOWLEDGEMENTS

Some of the work in this review was supported in part by the NIH National Insti-tute for Allergy and Infectious Diseases, ICIDR (International Collaborations in Infectious Diseases Research) grant No. UO1AI026512, by the NIH Fogarty International Center GIDRT Training grant No. D43TW006578, and MARCE grant No. U54AI057168. Additional support came from the NIH Eunice KennedyShriver National Institute of Child Health and Human Development (NICHD: ApoE grant No. RO1HD053131) with co-funding from the NIH Office of Dietary Supplements (ODS). Dr. Bartelt was supported in part by the Research Training in Digestive Diseases grant No. 5T32 DK007769.

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